This invention, in the fields of electrophoresis, microscopy, spectroscopy and molecular biology, relates to methods for characterizing polymer molecules or the like, for example, observing and determining the size of individual particles and determining the weight distribution of a sample containing particles of varying size. More particularly, this invention involves the use of microscopy and/or spectroscopy in combination with spectroscopic methods to characterize particles, e.g., nucleic acids, such as by measuring their positional and conformational changes when they are subjected to an external force, such as a restriction enzyme digest and by measuring their length and diameter or radius.
Traditionally, the molecular weight distribution of a sample of particles has been determined by measuring the rate at which particles which are subjected to a perturbing force move through an appropriate medium, e.g., a medium which causes the particles to separate according to size. A mathematical relationship is calculated which relates the size of particles and their migration rate through a medium when a specified force is applied.
Sedimentation is a well-known technique for measuring particle size, but, when applied to polymers, this method is limited to molecules with a maximum size of about 50-100 kilobases (kb). Attempting to measure larger molecules by this technique would probably result in underestimation of molecular size, mainly because the sedimentation coefficient is sensitive to centrifuge speed. See Kavenoff et al., Cold Spring Harbor Symp. Quantit. Biol., 381 (1974)).
Another popular method of separating polymer particles by size is by gel electrophoresis (see, e.g., Freifelder, Physical Biochemistry, W. H. Freeman (1976), which is particularly useful for separating restriction digests. In brief, application of an electric field to an agarose or polyacrylamide gel in which polymer particles are dissolved causes the smaller particles to migrate through the gel at a faster rate than the larger particles. The molecular weight of the polymer in each band is calibrated by a comparison of the migration rate of an unknown substance with the mobility of polymer fragments of known length. The amount of polymer in each band can be estimated based upon the width and/or color intensity (optical density) of the stained band. However, this type of estimate is usually not very accurate.
Pulsed field electrophoresis, developed by the present inventor and described in U.S. Pat. No. 4,473,452, which is hereby entirely incorporated herein by reference, is an electrophoretic technique in which the separation of large DNA molecules in a gel is improved relative to separation using conventional electrophoresis. According to this technique, deliberately alternated electric fields are used to separate particles, rather than the continuous fields used in previously known electrophoretic methods. More particularly, particles are separated using electric fields of equal strength which are transverse to each other, which alternate between high and low intensities out of phase with each other at a frequency related to the mass of the particles. The forces move the particles in an overall direction transverse to the respective directions of the fields. It should be noted here that the term xe2x80x9ctransversexe2x80x9d as used herein is not limited to an angle of, or close to, 90xc2x0, but includes other substantial angles of intersection.
One of the most significant problems with determining the weight of molecules by indirect measurement techniques, such as those described above, is that the parameters which are directly measured, e.g., migration rate, are relatively insensitive to small differences in molecular size. Thus, a precise determination of particle size distribution is difficult to obtain. The lack of precision may particularly be a problem when biological polymer samples, which tend to be unstable and contain single molecules inches in length, are involved.
While some of the known methods of determining particle size distribution in a polydisperse sample provide better resolution than others, few, if any, of the previously known techniques provide resolution as high as is needed to distinguish between particles of nearly identical size. Gel permeation chromatography and sedimentation provide resolution of only about M1/2 (M=molecular weight). Standard agarose gel electrophoresis and polyacrylamide gel electrophoresis provide resolution varying as xe2x88x92log M. Pulsed electrophoretic techniques are effective for separating extraordinarily large molecules, but do not provide much better resolution than standard electrophoresis. Thus, the ability to distinguish between particles of similar size, for example, particles differing in length by a fraction of percent, is inaccurate and problematic using the above-described measurement techniques.
Particles of higher mass (i.e., up to approximately 600 kb) can be resolved using conventional gel electrophoresis by reducing the gel (e.g., polyarylamide) concentration to as low as 0.035% and reducing field strength. However, there are also problems with this method. Most notably, the dramatic reduction in gel concentration results in a gel which is mechanically unstable, and less sample can be loaded. An electrophoretic run to resolve very large DNA molecules using a reduced gel concentration and field strength may take a week or more to complete. Furthermore, a reduced gel concentration is not useful to separate molecules in a sample having a wide range of particle sizes, because separation of small molecules is not achieved. Thus, if a sample containing molecules having a wide range of sizes is to be separated, several electrophoretic runs may be needed, e.g., first, a separation of the larger molecules and then further separation of the smaller molecules.
Other particle measurement techniques known in the art are useful for sizing certain molecules which are present in a bulk sample, (e.g., the largest molecules in the sample, or the average molecular size) but are impractical for measuring many polymers of varying length in a given sample. The viscoelastic recoil technique, (see Kavenoff et al, xe2x80x9cChromosome-sized DNA molecules from Drosophila,xe2x80x9d Chromosoma 411 (1973)) which is well known in the art, involves stretching out coiled molecules in a solvent flow field (e.g., a field which is created when fluid is perturbed between two moving plates) and determining the time required for the largest molecule to return to a relaxed state. Relaxation time is measured by watching the rotation of a concentric rotor which moves during the time of relaxation. While this technique is quite precise in that sample determinations vary as M1.66 when applied to large DNA molecules, it is not useful for sizing molecules other than the largest molecule in the sample.
Using light scattering techniques, which are known in the art, (e.g., quasi-elastic light scattering), the size and shape of particles are determined by a Zimm plot, a data analysis method which is known in the art. With these techniques, size dependence varies as M1. Light scattering requires that the solution in which the molecules to be measured are placed is pure, that is, without dust or any other contamination, and it is therefore unsuitable for sizing a DNA sample. Furthermore, it is not useful for sizing molecules as large as many DNA molecules, and is useful only for determining the average weight of particles in a sample, not the weight distribution of a sample with particles of various sizes.
Yet another particle measuring technique which is known in the art for measuring individual molecules provides measurements of particle size having limited accuracy. The average size and shape of individual, relaxed DNA molecules has been determined by observing the molecules under a fluorescence microscope, and measuring the major and minor axes of molecules having a spherical or ellipsoid shape (see Yanagida et al, Cold Spring Harbor Symp. Quantit. Biol. 47, 177, (1983)). This technique is performed in a free solution, without perturbation of the molecules.
The movement of small DNA molecules during electrophoresis has been observed (see Smith et al. Science. 243203 (1989)). The methods disclosed in this publication are not suitable for observation of very large DNA molecules, and techniques for measuring molecules are not discussed.
Practical weight determinations of particles such as polymer molecules depend not only upon maximizing the size dependencies of the directly measured parameters, but also upon factors such as the amount of sample needed, the time required to complete an analysis, and the accuracy of measurements. Gel permeation chromatography can be time-consuming and requires a large amount of sample. Methods such as conventional gel electrophoresis can be relatively time-consuming, require moderate amounts of sample, and cannot size very large DNA molecules.
Molecular sizing is a fundamental operation that touches virtually every aspect of genomic analysis from DNA sequencing to size measurements of lower eucaryotic chromosomal DNAs. Molecular size, given in kilobases, can be translated into centimorgans for many organisms, and vice-versa; and gel electrophoresis is generally used to determine these sizes. The basics in nucleic acid sizing technology, as practiced by the typical molecular biologist, have not changed very much in the past decade. This is understandable considering the simplicity of gel electrophoresis and its capacity for parallel processing of multiple samples. The data obtained from gels are readily interpretable. Given the size of most genes, gel electrophoresis techniques adapt well to their analysis. From characterization of restriction digests to discernment of one base differences in sequencing ladders, gel electrophoresis is the method of choice for size analysis of DNAs. Even the outcome of PCR (Mullis, Methods in Enzymol, 155335-350 (1987)) reactions is frequently monitored by sizing analysis. Pulsed gel electrophoresis extends this coverage even further to include chromosomal DNAs from lower eucaryotes (Schwartz, Cold Spring Harbor Symp. Quant. Biol., 4789 (1983); Schwartz Cell, 3767 (1984); Carle, Nucleic Acids. Res. 125647 (1984); Chu, Science 2341582 (1986); Clark, et al., Science 2411203 (1988). Because pulsed electrophoresis can resolve very large DNA molecules, its application has simplified the mapping of large genomes and provided a necessary tool for creating large YACs (yeast artificial chromosomes) (Barlow, et al., Trends in Genetics 3167-177 (1987); Campbell, et al. Proc. Natl. Acad. Sci. 885744 (1991)). However, pulsed electrophoresis was developed more than 10 years ago (Schwartz, Cold Spring Harbor Symp. Quant. Biol. 4789 (1983) and Schwartz Cell, 3767 (1984)). The surprising lack of significant sizing advances is contrasted to progress made in understanding the molecular mechanisms of conventional and pulsed electrophoresis (Zimm, Quart. Rev. Biophys. 25171 (1992); Deutsch, Science 240992 (1988).
Although molecular size determination has not advanced significantly in this decade, another aspect of genomic analysis, DNA detection technology, has progressed to a remarkable extent. These developments have impacted on gel-based methodologies as well as on the field of cytogenetics. A driving force has been the Human Genome Initiative and its goals to characterize the human genome and the genomes of model organisms by extensive mapping and sequencing. The new goals aimed at analyzing entire large mammalian genomes include increasing accuracy and high throughput of DNA mapping and sequencing. The first round of needed advances has come in part from a combination of sophisticated image processing methods (Glazer, Nature 359859 (1992); Quesada, BioTechniques 10616 (1991); Mathies, Nature 359167 (1992)); new DNA detection techniques and new DNA labeling/imaging systems (Glazer, Proc. Natl. Acad. Sci. 873851 (1990); Beck, Nucleic Acids Res. 175115 (1989)). Automation of gel electrophoresis based technologies demands clear, relatively unambiguous detection systems for operator-free function (Lehrach, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. pp39-81 (1989); Larin, Proc. Natl. Acad. Sci., 884123 (1991)). Sophisticated computational methods can extract usable data automatically from difficult conditions. A good example of a fully integrated approach to mapping comes from the Cohen laboratory which has combined all of these technological approaches together with xe2x80x9cmega-YACsxe2x80x9d (Bellanne-Chantelot, et al., Cell 70L1059 (1992)) to maximally boost output to a dramatic extent, although with problems inherent in the fidelity of these YACs (Anderson, Science 2591684 (1993)).
Construction of physical maps for eucaryotic chromosomes is laborious and difficult, in part because many of the current methodologies for mapping and sequencing DNA were originally designed to analyze genes rather than genomes, so that at present there is a premium on automating procedures such as PCR and blot hybridizations (Chumakov, Nature 359380 (1992)). Two techniques have played a fundamental role in the process of ordering and sizing DNA sequences from eucaryotic chromosomes. Electrophoretic methods have the advantage of good size resolution, even for long chains, but require DNA in bulk amounts. Sources include genomic DNA or YACs (Burke, Science 236806 (1987)). Single molecule techniques, such as fluorescence in-situ hybridization or (FISH), utilize only a limited number of chromosomes (Manuelidis, J. Cell. Biol. 95L619-625 (1982)) but have not yet attained a sizing capability comparable to that of pulsed electrophoresis. Ideally, one would like to be able to combine the sizing power of electrophoresis with the intrinsic loci ordering capability of FISH in order to construct accurate restriction maps very rapidly.
All considered, the evolution of various physical and genetic techniques has enabled far more to be accomplished than expected toward creation of a complete, physical map of whole chromosomes and the entire human genome (Bellanne-Chantelot, et al., Cell 70L1059 (1992); Chumakov, et al., Science (1992); Mandel, et al., Science 258103 (1992)). Despite this progress the situation can be improved in the following areas.
For fingerprinting YACs, chromosomal DNA is digested with several enzymes and then blotted and sometimes hybridized with several different repetitive sequences (Bellanne-Chantelot, et al., Cell 70L1059 (1992); Stallings, et al., Proc. Natl. Acad. Sci. 876218 (1990); Ross, et al., Techniques for the Analysis of Complex Genomes, Academic Press, Inc., San Diego, Calif., (1992)). Here, electrophoresis is used to size restriction fragments that are specifically identified by hybridization. The data density available for such an analysis is relatively low. For example, it is difficult to discern more than 100 bands in a given land in a typical agarose gel. Additionally, restriction fragments that are the same size cannot be resolved from each other and can only be discerned by careful, differential hybridization. Therefore, the fingerprint does not report nearly as much information as what would result if an ordered restriction map were to be made with the same enzyme(s) or even an accurate histogram of the size population. Such a histogram can only be obtained from gels by difficult measurements of band fluorescence intensities.
Gels are time-consuming. It takes time and care to pour gels and minutes to days to run, and it can take several days to do Southern analysis, although gels offer the opportunity for parallel sample analysis and, with multiplexing techniques (Church, et al., Science 240185 (1988)), this tremendous ability is probably maximized, sizing results are often difficult to digitize and to automatically tabulate.
Electrophoretic size resolution for commonly run agarose gels rarely exceeds mass. Although under limited conditions greater size resolution can be obtained (Calladine, Journal of Molecular Biology 221981 (1991)). Greater size resolution would enable simpler fingerprints with a higher information content. Although pulsed electrophoresis techniques can, under certain circumstances, boost size resolution, these results can be hard to interpret except in very narrow size ranges. Ultimately, these measured sizes are dependent on size markers which are limited in range for very large DNA molecules. For pulsed electrophoresis, the determined size is frequently inadequately interpolated between several size markers.
(iv) Usable sensitivity is limited to the subpicogram range except by exotic techniques (Glazer, et al., Nature 359859 (1992); Quesada, BioTechniques 10616 (1991)). However, now common phosphor imager systems have improved sensitivity some and make quantitation easier. The usable sensitivity range will dictate the type of sample that can be analyzed. For example, single-copy mammalian genomic hybridizations can be challenging to a novice. Mapping of end-labeled partial digestion of genomic DNAs is often not successful because of loss of attending sensitivity (Smith, et al., Nucleic Acids Res. 32387 (1976)) so that extensive analysis is difficult to do with genomic DNA samples. This necessitates the reliance on cloned genomic material, despite their limitations including problems with uncloneable regions, rearrangements and deletions. Although YACs enable cloning of such large genomic fragments and have served as the basis for many mapping approaches, they are not perfect mapping reagents and must therefore be used with great caution (Anderson, Science 2591684 (1993); Vollrath, et al., Science 25852 (1992); Foote, et al., Science 25860 (1992)).
Citation of documents herein is not intended as an admission that any of the documents cited herein is pertinent prior art, or an admission that the cited documents are considered material to the patentability of the claims of the present application. All statements as to the date or representations as to the contents of these documents are based on the information available to the applicant and does not constitute any admission as to the correctness of the dates or contents of these documents.
The present invention provides methods for physically characterizing large or small molecules, including polymers and particles, for determining molecule size, molecular weight, size distribution, weight distribution and/or enzyme cleavage maps of a homogenous, heterogenous, or polydisperse or varying sample of molecules.
This invention can determine at least one of molecule size, weight and cleavage or restriction maps using faster and more efficient methods, which also can provide better resolution, than methods known in the related art.
This invention also can size one or more particles or molecules using an extremely sensitive method, e.g., one which can use an amount of sample, e.g., as small as a few molecules or a single molecule.
Accurate size information for a polydisperse sample containing molecules having a wide range of sizes, is also provided, as well as providing this information more quickly than by using previously known techniques.
Methods of the present invention involve characterizing individual molecules, including deformable and non-deformable molecules, in a polydisperse sample by placing the molecules in a medium, applying an external force to the molecules, thereby causing physical changes (particularly conformational and/or positional changes), and then observing and measuring these changes. This method is useful for characterizing molecules of a variety of sizes, including the smallest molecules which are detected by a suitable microscope (the microscope optionally may be attached to a spectroscopic apparatus and thus molecules too small to be visualized may still be detected), and large polymers, which may be up to several or many inches in length when stretched to a linear conformation. Shear sensitive molecules (e.g., large molecules), which cannot be placed on a microscope slide without breaking when conventional techniques are used, are measured according to this invention by collapsing (condensing) the molecules before they are placed in the medium and then uncollapsing them after placement in the medium. This invention is useful for characterizing many types of particles which can be visualized or detected under a light microscope. Several non-limiting examples include polysaccharides, polypeptides, proteins, and nucleic acids (e.g., DNA or RNA).
Deformable molecules are particles or molecules which have a tendency to change conformation (shape), as well as position, when they are subjected to an external force. Non-deformable particles or molecules tend to have a substantially stable conformation even when subjected to an external force, but may undergo changes in position. Deformable molecules are usually reversibly deformable, e.g., they change conformation when an external force is applied, and then return to a configuration comparable to their original shape when application of the force is terminated.
This invention is particularly useful for measuring polymer molecules which are folded, coiled and/or supercoiled and are subject to conformational changes such as stretching, bending, twisting, contracting, and the like, as well as positional changes such as rotating, translating and the like. This invention is particularly useful when an external force is applied to molecules which are in some type of medium. However, if a free solution is used, application of an external force may not be needed to cause the molecules to change conformation or position.
Molecules which are large enough to be seen using a microscope are measured by visualization, e.g., by direct observation of a microscopic image. Particles may, alternatively, be measured using microscopy combined with any suitable spectroscopic technique, particularly if the particles are too small to be imaged (viewed with acceptable resolution).
Several non-limiting examples of useful spectroscopic methods include using polarized radiation as generated by a laser combined with measurement of refractive index or fluorescence dichroism, or using sensitive video cameras such as cooled charged coupled devices, silicon intensified target devices, and micro-channel plate detectors.
Samples containing a mixture of both small and large molecules, for example, small and large DNA molecules including chromosomes, are sized rapidly, with each molecule in the sample being measured simultaneously. The method of this invention involves measuring conformational and positional changes of individual, discrete molecules (or other particles), as contrasted to methods known in the art, which characterize a sample in bulk. The method of this invention may be applied to measure any number of molecules, ranging from a single molecule to a large number of molecules. If a sample containing a large number of molecules is measured, the number of molecules which are observed at one time will depend in part upon the field of view of the microscope and the extent to which the molecules are separated from each other. Viewing discrete, individual molecules, or measuring their role of relaxation after applying an external force permits complete deconvolution or separation of measured parameters.
The medium used in this invention is any suitable material. Preferably the medium will hold relaxed molecules in a relatively stationary position and yet permit movement of molecules which are subjected to an external force. However, a free solution also may be used. For measurements of molecular movement, a suitable medium is any medium which will permit different molecules to change conformation and position at different rates, depending upon their size, and perhaps upon their chemical composition.
For many uses of this invention, the preferred medium is a gel or a liquid. Preferably, the medium is anticonvective, but this is not absolutely necessary. The medium may or may not be inert. The choice of an appropriate medium will depend in part upon the size of the molecules which are measured, the tendency for the molecules to change position and shape, and the desired precision of the measurements. For example, when large molecules (or other molecules of similar size) are measured, a gel with a large pore size is preferably used.
The external force applied to the molecules is any force which causes the nondeformable or deformable molecules to undergo changes in conformation or position. For example, the force may be an electric field, solvent flow field, or a magnetic field, but is not limited to these types. The force may vary in direction, duration and intensity. A particularly useful way to perturb the molecules is by using electrophoresis, or by site specific enzyme digest, e.g., restriction enzyme digest of a DNA molecule.
The types of changes which are measured in this invention primarily include changes in conformation or shape, including stretching and relaxation rates, as well as length and diameter (or radius) measurements, and changes in position, including changes in orientation and rotation as well as translation within the medium. Molecules may undergo changes in conformation or position, or both. Different types of changes are measured according to various embodiments of the invention.
The techniques for measuring conformational and positional changes include, but are not necessarily limited to, microscopy (alone), and microscopy combined with spectroscopy. Several non-limiting examples of useful spectroscopic techniques include birefringence, linear or circular dichroism, and detection of fluorescence intensity.
Molecules which are large enough to be seen under a microscope can be measured by visualizing (imaging) the molecules. As non-limiting examples, a light microscope or a scanning/tunneling microscope may be used. While molecules may be viewed directly, it is useful to link the microscope to a low light sensitive video camera, connected to a computerized image processor (e.g., as described herein or as would be suggested to one skilled in the relevant arts) which records a series of photographs, even a motion picture, by digitizing or recording the images which are received. The image processor may itself comprise a computer, or may be linked to a computer which processes data based upon the images. Use of a computerized apparatus enables the movement of each individual molecule to be measured simultaneously. Furthermore, the relationship of molecules to one another may be detected, and several different parameters of a single molecule can be measured simultaneously.
Optionally, the microscope and image processor are connected to a spectroscopic apparatus. This technique is particularly useful for molecules which are too small to be visualized, but is also useful for sizing larger molecules as well.
In order to transform measurements of change in conformation and position into size measurements, it is generally necessary to generate (or otherwise obtain) data relating to physical changes of molecules of known size when the molecules are subject to external forces. xe2x80x9cMarkersxe2x80x9d are developed by measuring the parameters of molecules with known values of molecular weight. This information may be input into a computer or data processor in order to establish a relation between molecular weight and particular conformational and positional changes which are measured. Preferably, the markers are molecules of similar chemical structure to the molecules of unknown size (e.g., both molecules contain the similar chemical components), because rates of relaxation, reorientation and rotation may be dependent upon molecule composition. However, this may depend upon one or more other variables, e.g., polymer size, composition, molecular weight, pKa, amino acid or nucleic acid sequence, etc., and thus it may not always be necessary for the xe2x80x9cmarkersxe2x80x9d to have a composition similar to that of the molecules of unknown size.
Shear sensitive molecules are molecules which are subject to breaking when they are placed on a microscope slide using conventional methods. According to another aspect of this invention, such molecules may be collapsed into a higher density conformation before they are placed in a medium, in order to prevent breakage when the molecules are mounted on a microscope slide. Once they have been placed in the medium, they can be uncollapsed and measured by the same methods as the smaller molecules.
In one embodiment of the present invention, fluorescently stained, deformable molecules which are coiled, folded or otherwise configured in a native relaxed, folded or complexed conformation are placed in a medium and are temporarily deformed, related, unfolded, separated, cleaved or stretched by applying an external force. When application of the force is stopped, the relaxation or reversion time of the molecules (e.g., the time required for the molecules to return to their original, native state) is determined by direct microscopic observation of molecular movement or change, or by at least one of microscopy and spectroscopy. Alternatively, the kinetics of stretching are measured by following the stretching of the molecule after initiation of the external force. Rate measurements are calculated in various ways, for example, by determining an amount of change per unit time. Rates of change for molecules of unknown size are determined based upon rates of molecules of known size, such as by interpolation or extrapolation.
As, e.g., with the viscoelastic measurement technique known in the art, the relaxation time of molecules in a liquid according to this embodiment varies as about M1.66. In a gel, it is believed that resolution may be as high as M2-4. This is based upon known theoretical principles which show that molecules reptate in gels or confining matrices, and their relaxation time is much greater in a gel than in a solution (see, e.g., DeGennes, P. E., Scaling Concepts in Polymer Physics, Cornell University Press, N.Y. (1979)).
In a second embodiment, the reorientation time of a deformable or non-deformable molecule is measured. When molecules are first subjected to a perturbing force in one direction, and the direction of the perturbing force is then changed, for example, by 90xc2x0 or other transverse angle, such as 10-90xc2x0, 20-90xc2x0, 30-90xc2x0, 40-90xc2x0, 50-90xc2x0 or any range or value therein, small molecules quickly reorient themselves and start a new migration along the new path. Larger molecules, on the other hand, remain substantially immobile until they are reoriented in the direction of the electric field. Then, they too begin to move in the new direction. By that time, the smaller molecules will have moved ahead. Measurements of the rate at which the position of a molecule changes with respect to an external force may be measured, for example, by measuring changes in position (e.g., lateral and/or rotational movement) per unit time.
In a third embodiment, the rate at which a molecule rotates is determined when a series of external forces are applied. This method is particularly applicable to rod-shaped molecules, such as small DNA molecules, and elongated molecules which are maintained in a relatively uniform conformation. xe2x80x9cRotation timexe2x80x9d according to this invention is the amount of time required for a molecule to undergo a positional rotation of a particular angular increment, for example, 360xc2x0, when a particular set of external forces are applied.
By periodically switching pulse direction, intensity and length, molecules are caused to move slightly back and forth as they are rotated. This facilitates rotation, and is analogous to the way in which an automobile is manipulated into or out of a parallel parking space by alternating backward and forward motion. However, unlike an automobile, a rod-shaped or coil molecule may bend somewhat as it rotates. A pulsing routine may also function to keep a deformable molecule in a generally consistent conformation, in order to provide useful measurements, e.g., measurements which relate rotation time to molecular size.
Data for reorientation and/or rotation rates for molecules of known size may be used to develop a relationship between reorientation and/or rotation rate and molecular size, which then may be used to determine the size of various polymer molecules of similar composition and unknown size, such as those which are present in a polydisperse sample. Reorientation and rotation rate may be determined using microscopy (preferably combined with image processing) to directly observe positional changes, or by combining microscopy with spectroscopic measurements. Thus, these embodiments are useful not only for mid-sized and large molecules, but also for molecules that are too small to be imaged with acceptable resolution.
In yet another embodiment of this invention, the length of a molecule which has been placed in a medium is directly measured using microscopy. This technique provides direct measurement of the molecular size of any number of molecules. This method generally involves observing the curvilinear length of deformed molecules which are in a stretched state, e.g., during the application of an external force, or soon after termination of a force which has stretched a molecule. However, this method also may be applied to non-deformable molecules having an elongated shape, and measurement of such molecules does not require application of an external force before measurements are made. Preferably, this embodiment uses the same microscopy and imaging equipment as is described above.
In a fifth embodiment, the diameter (or radius) of molecules or other molecules suspended in a medium is measured. Application of a perturbing force is optional, because the diameter of a deformable molecule is preferably measured when the molecule is in a relaxed state, and the molecule is spherical, ellipsoidal or globular in shape. This embodiment may be used to measure molecules which are deformable or non-deformable, and involves the use of a light microscope attached to a computerized imaging device.
These five embodiments may be combined such that some or all of the above-mentioned parameters are measured simultaneously for one or more molecules.
A sixth embodiment of the invention is directed particularly to sizing very large molecules which tend to break if they are mounted on a microscope slide using conventional methods. In brief, this new technique involves collapsing the molecules before they are placed in the medium, using an agent which causes them to condense, and then uncollapsing the molecules after they have been placed in the medium. The molecules are then sized according to the method of embodiments one to five. The method for chemically collapsing molecules also may be used when it is desirable to place a large number of molecules in a small area, such as in microinjection, even if the molecules are not large or shear sensitive.
This invention provides a novel technique for mapping nucleic acid molecules. For example, when a nucleic acid is placed in a matrix and digested, the fragments are ordered by the computerized apparatus, and are sized by the methods described above. Thus, the order of the digests is quickly and accurately determined.
A further aspect of this invention provides for sequencing nucleic acid molecules by hybridizing probes to portions of a molecule. A nucleic acid is placed in a medium, to which suitable, desired probes are added. At least one recombinational enzyme may also be added. Reaction is initiated by an appropriate means, for example, the addition of ATP (adenosine triphosphate) and/or magnesium ions. After the probes have hybridized they are detected by the methods described above, namely, microscopy (alone) or microscopy in combination with spectroscopy.
Thus, the present invention provides an accurate method of determining the size of individual molecules and the weight distribution of a polydisperse sample of molecules. Another important advantage of this invention over the techniques of the prior art is that the measurable parameters for each molecule in a polydisperse sample, not just the largest molecule, are determined. Additional advantages are that (1) only one molecule is needed, and the sample may be very small, e.g., may consist of only one, or only a few molecules (2) measurements may be based on one representative molecule for each size in the sample, (3) the technique can be used for very large molecules (molecules too large to be measured by prior known methods), (4) data can be processed efficiently by computer, (5) measurements can be made more rapidly than methods known in the prior art (e.g. particularly as compared to slow electrophoresis processes, which may take several weeks), and (6) measurements are extremely accurate.
The present invention also can provide analysis of chromosomally sized DNA molecules and associated complexes utilizing new, ultra-rapid methodologies for determining the organization and structure of a eukaryote, such as an animal or human genome. Methodologies are provide which permit high resolution mapping for multiple individuals in a population of animals such as humans.
Such methods may involve optical mapping, which is a nonelectrophoretic approach, to rapidly create high-resolution ordered maps from chromosomally sized DNA molecules. Optical mapping produces ordered maps by fluorescently imaging single DNA molecules during restriction enzyme digestion. The resulting fragments are then sized by a number of single molecule methodologies according to the present invention. To facilitate mapping of mammalian genomes, optical mapping can be used to extend the size resolution to map fragments consisting of a few hundred base pairs, in addition to increase precision and throughput. To accomplish this, advanced intensity measurement techniques and sizing methodologies based on molecular relaxation can be used according to the present invention, as well as modified chamber designs and fixation techniques.
Detection Methods for Localization of Sequence Specific Sequences Including Hybridization to Single DNA Molecules. RecA protein-mediated hybridization approaches are also provided by the present invention, to precisely map sequences of large DNA by methods which may include optical mapping. Large target molecules can be imaged, localized and quantitated at specific sites via the visible gaps produced in the molecules at the hybridization site. Such sequence localization techniques can be combined in the present invention with sizing methodologies and sample handling techniques to improve throughput and versatility. Another method of the present invention is direct imaging of hybridization sites; this is based on conjugating RecA oligonucleotide filaments to different types of optically detectable tags allowing direct visualization of hybridization sites. Energy transfer techniques can also be used to enhance the specificity and effectiveness of tagged RecA-mediated hybridization as detected by imaging.
Increasing Throughput in mapping using Genomic DNA or YACs A number of single molecule methodologies can be combined in the present invention to dramatically increase the production of maps from YACs or genomic DNA. High throughput, flow-based optical mapping systems provided by this invention e.g., by producing high resolution, ordered restriction maps. RecA-assisted restriction endonuclease cleavage (RARE), according to this invention can selectively dissect genomes into large fragments. RARE and related techniques provide optical mapping approaches to rapidly map large genomic regions, without the need for cloning or sequencing.
Such optical mapping methodologies provide analysis methods for complex mammalian genomes, e.g., by applying sizing methodologies to raise the level of molecular size discrimination. These applications are facilitated by the discovery that relaxation phenomena of polymer molecules, e.g. DNA, demonstrate a remarkably high degree of size dependency. High throughput interfaces of methodologies of the present invention are also provided. The combination of high resolution and high throughput are preferably used with methods of this invention to provide high resolution maps of entire populations of a given species or animal subgroup. The value of cataloging high resolution maps of individuals for global genome comparisons is enormous, e.g., genetic analysis, and use in clinical settings for detecting complex heritable disorders, resulting from multi-genic determinants.
These and other advantages will become readily apparent from the detailed description which follows.